Glycolysis is required for mammalian sperm function and fertilization. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS), a unique isozyme expressed only during the late stages of spermatogenesis, appears to serve a pivotal role in regulating this metabolic pathway in spermatozoa. GAPDS has a novel N-terminus that tightly anchors it to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. GAPDS is more susceptible than somatic GAPD to inhibition by substrate analogs, which induce rapid and reversible male infertility. The long-term goal of this study is to identify mechanisms that regulate sperm glycolysis anc fertilization. The specific aims are to: 1) Determine if localization of GAPDS to the fibrous sheath is required for normal sperm function. These experiments will use knockout and transgenic mice to identify sequences required for GAPDS targeting and binding to this cytoskeletal structure, and to test the hypothesis that anchoring of GAPDS to the fibrous sheath is critical for normal sperm glycolysis, hyperactivated motility, and fertilization; 2) Identify protein interactions that anchor GAPDS to the fibrous sheath. The fibrous sheath, which serves as a scaffold for both glycolytic enzymes and protein kinase A subunits, is likely to be an important regulator of sperm motility. Proteins that interact with GAPDS will be identified by co-immunoprecipitation and yeast two-hybrid assays to better understand the formation and function of protein complexes along the fibrous sheath; 3) Determine if GAPDS plays a role in the assembly of sperm glycolytic enzymes or the phosphorylation cascade that occurs with hyperactivation. Because GAPDS is tightly bound to the fibrous sheath, it is well positioned to participate in the localization of other glycolytic enzymes to the principal piece of the sperm tail and in the regulation of hyperactivated motility. These studies will determine if GAPDS is required for the localization of other glycolytic enzymes to the principal piece, and if initiation of hyperactivated motility alters the activity, solubility, or phosphorylation status of GAPDS; 4) Identify key amino acids that are responsible for the novel enzymatic properties of GAPDS. Molecular modeling studies have identified eight amino acids near the substrate and cofactor binding sites of GAPDS that are different from somatic GAPD. Site-directed mutagenesis will be used to determine the effects of these amino acids on GAPDS activity and inhibition. These studies will determine if the human ortholog of GAPDS is a feasible target for developing highly specific male contraceptives, and if suppression of GAPDS activity by environmental and therapeutic agents could be a cause of infertility in men.